LITERATURE REVIEW
1.4 Structure of cationic peptides
Basically, all the cationic peptides have 2 distinguishing features, these are: (1) a net positive charge of a minimum of +2 due to the presence of arginine and lysine residues in the peptide sequences; and modified amino acids in the case of lantibiotics; (2) the peptides are folded into a 3-dimensional structure, consisting of a hydrophobic face consisting of non-polar amino acid side chains, and a hydrophilic face of polar and positively charged residues, i.e., amphipathicity. These peptides although similar in the above-mentioned features, shows considerable variation in length, amino acid sequence, and secondary structure. Most peptides possess these general characteristics, and on secondary structure fit into 4 major classes: a-helices, extended helices with the existence of a dominant
residue throughout the peptide, ~-sheetstructures stabilized by 2-3 disulfide bridges, and loop structures (Hancock, 1997) (Figure 1).
Some cationic peptides, have been found to differ somewhat from the defined structural classes, sometimes incorporating more than one structural class. Sapecin, the insect
defensin, contains 3 disulfide bonds, like animal defensins, but has a considerably different amino acid sequence and the presence of lysines and histidines as positively charged amino acids rather than lysines and arginines (Hanzawaet al., 1990). The solution structure of sapecin contains a flexible loop, an 8 amino acid a-helix, and 2 extended regions (Hanzawa et al., 1990). This is thought to be due to the different positions ofthe cysteine disulfide bonds in the animal and insect defensins. Contrary to this hypothesis, scorpion toxins with positions of disulfide bonds similar to animal defensins, have a structure comprising a short triple-stranded ~-sheetregion, together with a 9 amino acid a-helix (BontemsetaI., 1991).
1.4.1 Structure-Function relationships
Numerous synthetic peptides have been produced in order to become more acquainted with structure-function relationships of these antimicrobial peptides. A few governing principles about structure and function on the cationic antimicrobial peptides are: (1) a change in amino acid sequence of a peptide influences the activity in a very specific manner, e.g., each position of the 26 amino acid mellitin was individually omitted to generate a series of 24 omission analogs to test for differing bioactivities amongst the analogs (Blondelle and Houghten, 1991). Testing for hemolytic activity of these analogue peptides showed that the activities were nearly the same for all the peptides, except there was increased activity in the analog lacking isoleucine at position 20 (Blondelle and Houghten, 1991). Modellins are synthetic peptides of different lengths and
hydrophobicities (BessalleetaI., 1993). These amphipathic peptides were synthesized to be composed of 16-17 amino acids, a hydrophilic lysine composed face, and a highly hydrophobic face due to the tryptophan and phenylalanine residues with high antibacterial
and hemolytic activities (Bessalle et aI., 1993). Replacement oftryptophan and phenylalanine with leucine, showed reduced hydrophobicity of the modellins, and a resultant decrease in hemolytic activity was noted, as well as a slightly decreased bioactivity (Bessalleet aI., 1993); (2) for a-helical peptides, changes that increase the tendency to form an a-helix in aqueous solution tends to increase activity (Ohet aI., 1998;
Dykeset aI., 1998; Blondelle and Houghten,,1991; Cornutet al., 1994); (3) it seems that no relationship exists between the number of positive charges and activity, although the position bfspecific positive charges is important (Powellet al., 1995; Leeet al., 1997a);
(4) chirality of peptide structures have little significance (Wadeet al., 1990), and full activity can be achieved with peptides containing either all L- and all D-amino acids in their respective right handed or left-handed helical conformations (Merrifieldet aI., 1995);
(5) there is no absolute relationship between decreased lysis or binding to cells and its correlation with a decreased minimum inhibitory concentration (Hancock, 1995); and, (6) in peptides that contain cysteine disulfide bonds, a reduction of the disulfide bridges destroys activity (Hancocket aI., 1995).
Synthetic peptides are now being produced in large numbers and with various modifications of structure in order to produce peptides with improved activity and decreased hemolytic activity among other required features. Length of the peptide may have some influence on cationic peptide bioactivity. There is usually decreased activity in shorter peptides (Hancock and Chapple, 1999; Bessalleet aI., 1993).
Understanding the general principles that influence structure-activity relationships allows manipulation for enhanced activity. A novel cationic peptide, CP-ll, which is based on the structure of the bovine neutrophil peptide, indolicidin, was designed to increase the number of positively charged residues, maintain the 13 amino acid short length, and increase amphipathicity, with respect to these features of indolicidin (Falla and Hancock, 1997).
CP-ll and a carboxymethylated derivative CP-IIC, demonstrated enhanced activity against Gram negative bacteria, and the yeastCandida albicans, a reduced hemolytic activity, and maintained the same activity levels as indolicidin against staphylococci (Falla and Hancock, 1997).
1.4.2 Mode of action
There have been numerous studies to determine the mode of action of the cationic antimicrobial peptides, specifically the cecropins, magainins, mellitins and defensins (Hancock and Lehrer, 1998). Currently, the main site of action of these peptides is the cytoplasmic membrane (Fallaet al., 1996; Hancock, 1995; Matsuzaki et al., 1997). In Gram positive bacteria the interaction is only with one membrane, i.e., the cytoplasmic membrane. On the other hand, Gram negative bacterial species require, a more involved interaction due to the existence of an outer membrane, and a cytoplasmic membrane.
The predicted mechanism of action based on the model of Christensenet al (1988) (Figure 2), states that there is an initial electrostatic interaction between the negatively-charged phospholipid bilayer of the cytoplasmic membrane and the cationic peptide. The bacterial cytoplasmic membrane has a large electric potential, and this influences transition of peptides from an unstructured to structured form. There is an aggregation of peptidesina manner that results in the hydrophobic faces of the peptides being directed toward the interior of the membrane and their hydrophilic faces form the channel pointing inwards.
Insertion of these aggregated peptides into the cytoplasmic membrane resultsindestruction of membrane integrity, and subsequent bacterial cell death, caused by the leakage of cytoplasmic molecules. Factors that favour the formation of channels include, high negatively charged lipid composition of the phospholipid bilayer, large transmembrane potentials maintained by the proton motive force, a lack of cationic lipids and cholesterol (Christensenet al., 1988), which is characteristic of bacterial membranes (Hancock, 1997).
Eukaryotic membranes have cholesterol as part ofthe lipid composition of the cell's membrane, low membrane potentials and low anionic lipid content, and this could be an explanation for the cationic peptide's specificity for bacterial cell membranes (Boman, 1995).Analternative to the more accepted hypothesis is that the cationic antimicrobial peptides cluster at the cytoplasmic membrane surface and cause a cooperative
permeabilisation ofthe cytoplasmic membrane, also referred to as the 'carpet effect' (Boman, 1995).
Figure 1 Examples of structures of four classes of cationic peptides. (a) p-strand human defensin-l, (b)0:- helical cecropin mellitin hybrid, (c) extended coil indolicidin, (d) loop structure bactenecin. Positive charges denoted by(+), amino termini denoted by (N), and disulfide bridges are present in (a) and (d). In (a) the three
p-strands are denoted by pairs of one, two or three lines on the backbone (Hancock, 1997).
For Gram negative bacterial species it has been proposed that cationic antimicrobial peptides interact with the bacterial outer membrane lipopolysaccharide (LPS), and are taken up by a 'se1f- promoted uptake' pathway (Hancock, 1997; Hancocket aI., 1995;
Hancock and Lehrer, 1998). The LPS of the outer membrane of Gram negative bacteria is a highly anionic glycolipid. Initiation of the 'self-promoted uptake' pathway occurs when the cationic antimicrobial peptide, interacts with the anionic, divalent cation-binding sites on LPS. Ithas been reported that the addition of 2 positive charges on the carboxyl terminus of a cecropin-mellitin hybrid (CEME), resulted in an enhanced interaction of the peptide with LPS and consequently, the outer membrane (Piers and Hancock, 1994). Direct interactions of other cationic peptides like magainins and defensins with LPS have been demonstrated (Hancocket aI., 1995). These interactions seem to be several orders of magnitude higher than the normal divalent cations, demonstrating that this binding is quite
an efficient process. This interaction causes disruption to the nonnal outer membrane barrier properties by the production of transient 'cracks', which pennits the passage of various small molecules, like the cationic antimicrobial peptides , into the cell (Hancock, 1997; Piers and Hancock, 1994) (Figure 2). Itis assumed that peptides, that are specific for Gram positive bacteria, lack the ability to access the 'self-promoted uptake' pathway because of the absence of the outer membrane.
Mode of action studies done on lantibiotics show that unlike the amphipathic pore-fonning lantibiotics e.g., nisin (Sahl, 1991), the globular lantibiotics appear to be involved in inhibition of enzyme reactions (BrOtzet al., 1997). Studies done on the lantibiotic mersacidin, in particular, indicates no impainnent of the overall integrity of the cell membrane (Brotzet al., 1998), but instead it exerts bactericidal action by the inhibition of the transglycosylation level of peptidoglycan biosynthesis by the interaction of the Lipid II component of peptidoglycan (Brotz et al., 1998). The lantibiotic actagardine also shows similarity to mersacidin with respect to its mechanism of bactericidal activity (Brotzet al., 1998). Mersacidin and actagardine are of similar size and hydrophobicity, contain 4 intramolecular thioether bridges, and consequently the same globular structure (Brotzet al., 1997). The findings are significant in that there may be different mechanisms of action of different peptides depending on their structures, and that not all antimicrobial peptides have the same mode of action.